This invention relates to processes for the preparation of ansamitocins, in particular ansamitocins which can be converted to maytansinol.
Highly cytotoxic maytansinoid drugs and their therapeutic use have been described in U.S. Pat. No. 5,208,020. These drugs can be prepared from ansamitocin precursors produced by fermentation of microorganisms such as Actinosynnema.
Under defined culture conditions, Actinosynnema spp. such as Actinosynnema pretiosum produce a number of related ansamitocins. The major product is ansamitocin P-3 with an isobutyryl moiety at the C-3 position. Other ansamitocins differing only in the C-3 acyl side chain are produced as minor components, P-1 (ethionyl moiety), P-2 (propionyl moiety), P-3xe2x80x2 (butyryl moiety), P-4 (isovaleryl moiety) and P-4xe2x80x2 (valeryl moiety). All these compounds may undergo reductive cleavage to produce a common product, maytansinol (form P-0). In addition, a number of other ansamitocins are produced at low levels, which are modified at other sites in the molecule (hydroxylated or n-demethylated). These do not produce the desired P-0 on deacylation.
Processes for ansamitocin P-3 production from Actinosynemma spp. have been described in U.S. Pat. Nos. 4,162,940; 4,228,239; 4,356,265; and 4,450,234. In general, these methods require adding a filter aid and a water-miscible solvent to whole fermentation broth, removing solids and extracting the aqueous fraction with a water-immiscible solvent, concentrating and precipitating with petroleum ether, purifying the precipitate using silica chromatography, and crystallizing followed by further chromatography or re-crystallization. Alternative processes utilize Diaion HP-10 adsorption instead of silica chromatography followed by further solvent extraction and crystallization.
These processes can be used to gain acceptable yields of ansamitocin P-3, but the methods involve a large number of stages which introduce limitations in large-scale production operations, particularly because of the extremely toxic nature of the ansamitocin compounds and the necessity of ensuring the safety of the human operators of the processes. Thus, a need exists to have a safer alternative procedure available, utilizing fewer and more contained stages.
One aspect of the present invention is a method for preparing purified ansamitocins comprising the steps of:
a. culturing an ansamitocin-producing microorganism in a liquid culture medium;
b. treating the culture medium to facilitate solvent extaction of ansamitocins;
c. extracting ansamitocins from the culture medium with an aromatic hydrocarbon solvent;
e. concentrating the extracted ansamitocins; and
f. purifying the ansamitocins by crystallization.
Another aspect of the present invention is a method for preparing purified ansamitocins comprising the steps of:
a. culturing an ansamitocin-producing microorganism in a liquid culture medium;
b. extracting ansamitocins from the culture medium with an aromatic hydrocarbon solvent;
c. concentrating the extracted ansamitocins; and
d. purifying the ansamitocins by crystallization.